Naturally acquired tolerance and sensitization to minor histocompatibility antigens in healthy family members

Abstract

Bidirectional cell transfer during pregnancy frequently leads to postpartum persistence of allogeneic cells and alloimmune responses in both the mother and in her offspring. The life-long consequences of naturally acquired alloimmune reactivity are probably of importance for the outcome of allogeneic stem cell transplantation. We investigated the presence of CD8(pos) minor histocompatibility (H) antigen-specific cytotoxic T lymphocytes (T(CTL)) and CD8(pos) minor H antigen-specific T regulator cells (T(REG)) in peripheral blood cells obtained from 17 minor H antigen-disparate mother-offspring pairs. Absence of minor H antigen-specific T(REG), as marked by the feasibility to expand T(CTL) from isolated tetramer(pos) populations, was observed in 6 mothers and 1 son. The presence of minor H alloantigen-specific T(REG) was observed in 4 mothers and 5 sons. These T(REG) were detected within isolated tetramer(dim) staining fractions and functioned in a CTLA-4-dependent fashion. Our study indicates that both T(CTL) and T(REG) mediated alloimmunity against minor H antigens may be present in healthy female and male hematopoietic stem cell donors, potentially influencing graft-versus-host reactivity in different ways.

Figure 1

Bidirectional routes of familial exposure to minor H alloantigens. Pathway C illustrates exposure of the offspring to noninherited maternal minor H alloantigen HA-1^H after transfer of cells during pregnancy (indicated by arrow). Maternal exposure to fetal inherited paternal minor H alloantigen HY or HA-1^H is illustrated by pathway A and B, respectively.

Figure 2

Tetramer staining patterns and cytolytic function of CD8⁺ minor H antigen–specific T cells obtained from healthy women and men. (A,C) Representative tetramer-binding profiles of HY-specific T cells (A) and HA-1–specific T cells (C) detected ex vivo in CD8-enriched PBMCs after nonstringent FACS sorting. The rectangle indicates the total tetramer⁺ population isolated during a second round of FACS sorting. (B,D) The cytolytic activity, indicated as percentage lysis on the y-axis, of polyclonally expanded tetramer⁺ fractions tested against various target cells: □ indicates HLA-A2⁺, HA-1⁻ female target cells; ■, HLA-A2⁺, HA-1⁻ female target cells pulsed with HY peptide (B) or HLA-A2⁺, HA-1⁻ female target cells pulsed with HA-1 peptide (D); and ●, HLA-A2⁺ HA-1⁺ male target cells. The effector:target cell ratios (as depicted on the x-axis) were calculated according to the corresponding percentage of HY^A2 tetramer-binding T cells as shown in the center plots.

Figure 3

Presence or absence of minor H antigen–specific regulatory T cells analyzed in the tvDTH assay. Total PBMCs of 2 mothers with male offspring were tested for minor H antigen HY-driven bystander suppression of recall responses. PBMCs were injected together with either a mixture of recall antigens comprising tetanus toxoid (TT) and diphtheria toxoid (D), minor H allopeptide HY (allo HY) or minor H self-peptide HA-2^V or HA-2^M (self HA-2^V, self-HA-2^M) alone or with a combination of TT/D + allo- or self-peptide into the footpads of CB17.SCID mice. Footpad swelling, indicated as net swelling on the y-axis, was measured 24 hours later. Percentages indicate the percentage of inhibition of the recall response (■) in the presence of allo- (▨) or self-(□) minor H peptide. Mother ♀4 (left graph) displays a tvDTH regulator phenotype; mother ♀7 (right graph) is classified as a tvDTH nonregulator.

Figure 4

Dissection of HLA-A2/minor H peptide tetramer staining profiles. (A,C) Definition of tetramer^bright and tetramer^dim gate settings (as indicated by dotted lines) using a tetramer^bright staining cytolytic HY (A) or HA-1 (C) specific T-cell clone titrated into CD8-enriched PBMCs. (B) Analysis of tetramer-binding profiles of 2 female donors (♀9 and ♀6) with male offspring after nonstringent sorting of HY^A2 tetramer⁺ CD8⁺ cells. (D) Analysis of HA-1^A2 tetramer-binding profiles of 2 HA-1^R/R male donors (♂1 and ♂2) with a HA-1^H/R mother after nonstringent sorting of HA-1^A2 tetramer⁺ CD8⁺ cells. The solid box represents the total CD8⁺ tetramer⁺ population; tetramer^bright staining T cells are plotted in black; tetramer^dim staining T cells are plotted in gray. Percentages indicate the distribution of each T-cell subset in the total tetramer⁺ fraction.

Figure 5

Phenotypic and functional analysis of minor H alloantigen–specific T_REG. Results from 1 male donor (♂5) and 2 female donors (♀1 and ♀6) with minor H antigen disparate family members are shown. (A) Cell-surface expression of CD25 and CTLA-4 by CD8⁺ HA-1^A2 (top graph) or HY^A2 (center and bottom graphs) tetramer^dim staining T cells (indicated by circle) isolated by 2 consecutive rounds of FACS sorting from CD8-enriched PBMCs. (B) Defining the role of CTLA-4 in minor H alloantigen–driven bystander suppression of recall responses. Total PBMCs, obtained from the same blood sample as shown in panel A, were injected together with the recall antigens tetanus toxoid (TT) and diphtheria toxoid (D), allopeptide (HA-1 for ♂5; HY for ♀1 and ♀6) alone or a combination thereof into the footpads of CB17.SCID mice. Footpad swelling indicated as net swelling is depicted on the y-axis. Uncovering of recall antigen–induced footpad swelling was induced by coinjection of blocking CTLA-4 antibodies (αCTLA-4) or an isotype control antibody (iso). The percentages indicate the percentage of inhibition of the recall response (■) when minor H allopeptide is coinjected with or without blocking CTLA-4 antibody.